PROJECT SUMMARY/ABSTRACT Bacteria prevent viral infection by deploying CRISPR-Cas immunity, which features RNA-guided nucleases that recognize and cleave phage genomes with sequence specificity. Our understanding of the mechanisms and applications for these systems has advanced dramatically in recent years, however, our appreciation for the natural physiology of CRISPR-Cas interactions with phages is lacking. This proposal focuses on the discovery, characterization and evolution of the phage counter-response to CRISPR-Cas immunity. My lab has recently discovered ?anti-CRISPR? proteins produced by Listeria monocytogenes phages that inhibit CRISPR-Cas9 function through distinct mechanisms. While three of the proteins (AcrIIA2-4) interact directly with the Cas9 RNA- guided nuclease, AcrIIA1 functions in the absence of such an interaction. Moreover, acrIIA1 is the most widespread anti-CRISPR gene discovered to date, encoded by phages, non-phage mobile elements, and core genomes across the Firmicutes phylum. Preliminary evidence suggests that this protein represses the accumulation of Cas9 protein in the cell, suggesting a regulatory role towards biogenesis inhibition. No such regulatory protein has been previously described. AcrIIA1 possesses a predicted helix-turn-helix domain, which suggests a mechanism that may involve nucleic acid interactions. Interestingly, phages that infect L. monocytogenes do not possess just one anti-CRISPR gene, they often encode AcrIIA1, in addition to at least one of the inhibitor proteins (AcrIIA2-4). The functional importance of this apparent `multi-pronged' CRISPR- Cas9 attack is unknown. First, we will design isogenic phages to determine the contribution of multiple anti- CRISPRs to phage fitness, during lytic replication and lysogeny (phage integration). Second, we will determine whether AcrIIA1 makes direct interactions with any CRISPR-Cas9 promoter elements or RNA transcripts to interrogate its mechanism of action. Unbiased interaction profiling will also be conducted to fully capture AcrIIA1 biology. Lastly, given how widespread acrIIA1 homologs are, we will conduct comprehensive bioinformatics to determine the evolutionary origins of this protein superfamily and identify essential residues for function. Preliminary analyses have revealed an acrIIA1 homolog is found adjacent to a CRISPR-Cas9 operon in Lactobacillus, suggesting a functional linkage between acrIIA1 and endogenous CRISPR-Cas9 regulation. Additionally, we will utilize acrIIA1 as an anti-CRISPR marker to facilitate new anti-CRISPR discovery. This will contribute to our ultimate goal; identifying all CRISPR-Cas systems that are inhibited by phage anti-CRISPR systems. Additionally, CRISPR-Cas9 inhibitors provide new contributions to the gene editing toolbox, as a means to enact post-translational inactivation and limit off-target gene editing. Taken together, I propose that AcrIIA1 is a widespread CRISPR-Cas regulatory protein that bacteria and phage possess. We will determine its role, mechanism, and diverse reach, which will vastly expand our understanding of CRISPR-Cas biology, phage- host interactions, and contribute new reagents for CRISPR-Cas applications.